Summary

● Because bacteriophages provide insights into complex phenomena, they are attractive subjects for research.
● Phage gene products that prevent or eliminate bacterial infections could provide alternative strategies for fighting antibiotic-resistant pathogens.
● Bacteriophage T7 overcomes host obstacles at every step of its lytic cycle, adapting to changes in host receptors, evading restriction enzymes, generating needed nucleotides, and inactivating host enzymes that interfere with its propagation.
● Studying how bacteriophage T7 interacts with its host expands our understanding of how viruses or other microorganisms interact with even more complex host organisms.

The abundance of phages and their importance to evolution and to ecology provide an incentive to study them. The golden era for studying phages stretched from the 1920s through the late 1980s, when the relative simplicity of their replication cycle proved critical for learning fundamental biology, including identifying the hereditary role of DNA and uncovering the nature of the genetic code.

During the 1990s and until recently, phage biology fell into relative neglect. However, bioinformatics and resources such as bacterial knockout collections and open reading frame (ORF) libraries are reviving the field, and it again is bringing important payoffs. For example, researchers recently elucidated the CRISPR system as a bacterial defense mechanism against phages (Microbe, May 2009, p. 224).

Moreover, there is renewed interest in phages as therapeutic agents against bacterial infections, reflecting, in part, frustrations over the emerging resistance of bacteria to conventional antibiotics. Thus, for example, in the country of Georgia, physicians are using phages to treat infections. Although phage-based treatments of patients are not authorized in the United States, the Food and Drug Administration recently approved the use of a phage mixture to use on particular foods to prevent them becoming contaminated with Listeria.

Bacteriophage T7 and Its Escherichia coli Host

Phage T7 depends on its bacterial host Escherichia coli to propagate. A great deal is known about this host-viral partnership, including the tentative roles of more than half of each of the 56 genes of T7 and the 4,453 genes of E. coli.

T7, an obligatory lytic phage, unleashes more than 100 progeny phage per host in less than 25 min under optimal conditions. The 39,937-bp, double-stranded DNA genome of T7 is transcribed from left to right, with each gene on the physical map sequentially numbered (Fig. 1). The essential genes are assigned integral numbers, while the nonessential genes are assigned noninteger numbers reflecting their relative positions between essential genes. Genes 2.5, 6.7, and 7.3 are essential gene exceptions to that naming practice, as is gene 7, which is now considered nonessential. Class I genes, expressed early in infection, establish favorable conditions for phage growth, class II genes are expressed later and mainly encode DNA replication proteins, and class III genes are expressed during the late stages of phage growth and mainly encode structural gene products.

Overcoming Host Obstacles to Bacteriophage T7 Propagation

The lytic cycle of T7 phage is divided into several steps, including adsorption, DNA penetration, DNA replication, and DNA packaging (Fig. 2). During adsorption, T7 attaches its tail fiber proteins to lipopolysaccharide molecules (LPS) on the outer membrane of a host cell. Loss of function of any of the nine nonessential genes in the LPS corebiosynthesis pathway of the host confers resistance to T7 infection, and the frequency of these mutations in the laboratory is about 10-5. However, T7 phage can overcome this resistance by acquiring mutations in genes encoding its tail proteins. In fact, by selecting for T7 phages that infect LPS mutants, we isolated T7 phages that adsorb to the host in a LPS-independent manner. These mutants extend their host range about 200-fold compared to wild-type T7.

The mainstay of resistance to phage is the bacterial restriction system, which recognizes and cleaves specific DNA sequences. By having underrepresented recognition sequences, T7 phage evades restriction enzymes, particularly the type II restriction systems. The phage genome also encodes gene product (gp) 0.3, a protein that mimics the structure of a DNA molecule and specifically binds to and inactivates the type I restriction enzyme. As an additional protective measure, the DNA sequence encoding gene 0.3 lacks any sequence recognized by the type I restriction system. Further, the sequences located toward the middle and end of the T7 genome enter the cell only after gene 0.3 is expressed at a sufficient level to protect them. Meanwhile, how T7 avoids the host RecBCD nuclease complex that degrades linear DNA remains a mystery. The phage encodes gp5.9 that specifically binds and inhibits the nuclease activity of RecBCD nuclease. However, gene 5.9 is not expressed until after T7 DNA enters a host cell.

The clustered, regularly interspaced short palindromic repeats (CRISPR) system is another way in which host cells defend against bacteriophages and other extrachromosomal elements. No phage gene product is known to inhibit the CRISPR system. However, we determined that the T7 protein kinase phosphorylates threonine and serine residues of one of the components of the CRISPR system, CasB. Moreover, when this T7 protein kinase phosphorylates several enzymes, including the host RNA polymerase and RNaseE, it inhibits them, whereas it enhances the activity of other enzymes, including E. coli RNase III. We are testing whether gp0.7 phosphorylation of CasB is a phage counter-defense against CRISPR.

The rapid synthesis of T7DNAto yield 100 progeny phage necessitates access to a large pool of nucleoside 5′-triphophates, which is a complex synthetic pathway within host cells (Fig. 3). More than 80% of nucleotides used in synthesizing the DNA of T7 progeny derive from the host chromosome. T7 achieves this efficient utilization of nucleosides by encoding an endonuclease, gp3, and an exonuclease, gp6, that together degrade the host chromosome into nucleoside 5′- monophosphates. These nucleoside monophosphates must be phosphorylated to yield nucleoside 5′-triphosphate substrates for T7 DNA polymerase. These phosphorylations are carried out by both host and phage enzymes. For example, T7 gp1.7 is a nucleotide kinase that phosphorylates both dGMP and dTMP. Its activity was identified through disruptive mutations in a genetic screen to isolate T7 DNA polymerase mutants that are protected against the chain-terminating dideoxthymidine triphosphate (ddTTP).

In another screen, we found that the host cmk gene product, dCMP/CMP kinase, is essential for T7-but not host cell- growth. Thus, while T7 phage encodes a dGMP/ dTMP kinase, it does not encode a dCMP kinase. Because dCTP can be synthesized from UTP via another pathway, the cmk product is dispensable for the host but not T7, presumably due to its faster metabolism. Another explanation for the growth deficiency of T7 phage in the absence of the cmk gene product is the reduced pool of rCTP. Indeed, we find that a specific mutation in T7 primase partially overcomes this growth deficiency on cmk cells. T7 primase makes primers that are rich in cytosine, and ongoing experiments suggest that a mutant primase is less restricted to using CTP for primers than is the wild-type enzyme.

The dNDP molecules are phosphorylated to the corresponding dNTPs by the abundant ndk gene product, a broad-acting nucleoside diphosphate kinase. The expanded pool of nucleoside triphosphates in a cell following T7 phage infection is unusual for the host. In fact, dgt of E. coli encodes a dGTPase that hydrolyzes dGTP to guanosine and tripolyphosphate, thus maintaining a balanced pool of dGTP. An absence of this dGTPase yields a mutator phenotype. T7 phage, which requires high levels of dGTP, encodes gp1.2, which specifically inhibits this host enzyme. However, gp1.2 is essential for phage growth only when dgt is overexpressed. For example, a dgt promoter mutation results in increased dgt expression, restricting phage growth in the absence of gp1.2. Even though gp1.2 activity is dispensable for phage growth when dgt is expressed at normal levels, its presence presumably helps maintain levels of dGTP in the host cell that are sufficient for phage propagation.

Packaging of Progeny T7 DNA Is a Complicated Process

Packaging T7 DNA is a complicated process during which a concatemeric T7 DNA molecule is converted into unit lengths, each of which is then inserted into an individual capsid. The T7 RNA polymerase (RNAP) recognizes the genomic end of each separate unit of the concatemer, presumably pausing the elongation complex at a unique site located immediately after the concatemeric junction. The role of T7 RNAP as a signaling molecule marking the genomic end upon pause in transcription provides several advantages to the phage. First, the T7 RNAP elongation complex selects only T7 DNA, excluding remnants of host DNA. Second, the requirement for elongation complex prevents stochastic packaging at different T7 promoters. Third, pausing dictates specificity for the packaging site of the T7 genome. Because the host RNAP could cleave T7 DNA near its early host promoters, T7 inactivates that host enzyme during the late stages of the T7 lytic cycle. The T7 gene 2 product, gp2, which is produced early during infection, binds to the β′ subunit of the host RNAP and prevents its loading onto RNA polymerase promoters near the left end of the T7 genome. Furthermore, T7 gp0.7 phosphorylates the β′ subunit of the host RNAP, increasing its transcription termination. We find that when the host gene, udk, is overexpressed, T7 gp2 no longer inactivates the host RNAP to a sufficient extent, leading to premature breakage of T7 DNA and lack of phage growth.

According to our model (Fig. 4), T7 DNA is cleaved due to the host RNAP causing a roadblock to the faster T7 RNAP. This accidental pause recruits the DNA packaging machinery, which begins to cleave the DNA. Ordinarily, these steps occur only at the unique pause site of the T7 RNAP, a site that is immediately downstream of the concatemer junction where it generates genomic ends. The accidental pauses followed by recruitment of the packaging machinery, however, damage the phage DNA, especially near the host promoter sites. Several lines of evidence support this model. First, the host RNAP destroys T7 DNA, while deleting the early host promoters alleviates the requirement to inactivate the host RNAP. Second, in the absence of T7 DNA packaging proteins such as gp19 and gp10, the phage DNA remains intact despite host RNAP activity, indicating that packaging proteins can mediate DNA cleavage. Third, mutations in gp3.5 that reduce the pausing of T7 RNAP also alleviate the requirement to inactivate the host RNAP. Thus, pausing leads to cleavage of the T7 DNA in the absence of sufficient host RNAP inactivation.

Outlook for T7 Phage Research

Despite a wealth of data on T7 phage structure, genetic organization, timing of gene expression, and each step of its lytic cycle, there is still much to learn about this relatively simple virus. For example, we understand the function of only half the genes of T7 phage. Protein-protein interactions with the host and with other phage proteins also remain obscure. Identifying these functions and interactions may reveal new mechanisms of gene regulation and of host response against viral attacks.

In addition, its fast growth rate and rapid adaptivity make T7 phage well suited for exploring evolutionary principles. Not only do such studies complement biochemical and genetic research on this phage, they also could lead to a better understanding of viral resistance to inhibitors and to new approaches for developing tools to fight antibiotic-resistant pathogens.

SUGGESTED READING

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Gawel, D., M. D. Hamilton, and R. M. Schaaper. 2008. A novel mutator of Escherichia coli carrying a defect in the dgt gene, encoding a dGTP triphosphohydrolase. J. Bacteriol. 190:6931-6939.

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Qimron, U., B. Marintcheva, S. Tabor, and C. C. Richardson. 2006. Genome-wide screens for Escherichia coli genes affecting growth of T7 bacteriophage. Proc. Natl. Acad. Sci. USA 103:19039-19044.

Zhang, X., and F. W. Studier. 2004. Multiple roles of T7 RNA polymerase and T7 lysozyme during bacteriophage T7 infection. J. Mol. Biol. 340:707-730.